Medicago truncatula as a Model for Dicot Cell Wall Development

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Bioenerg. Res. (2009) 2:59–76 DOI 10.1007/s12155-009-9034-1

Medicago truncatula as a Model for Dicot Cell Wall Development Mesfin Tesfaye & S. Samuel Yang & JoAnn F. S. Lamb & Hans-Joachim G. Jung & Deborah A. Samac & Carroll P. Vance & John W. Gronwald & Kathryn A. VandenBosch

Published online: 7 May 2009 # The Author(s) 2009. This article is published with open access at

Abstract We have initiated a genome-wide transcript profiling study using the model legume Medicago truncatula to identify putative genes related to cell wall biosynthesis and regulatory function in legumes. We used the GeneChip® Medicago Genome Array to compare transcript abundance in elongating versus postelongation stem internode segments of two M. truncatula accessions and two Medicago sativa (alfalfa) clones with contrasting stem cell wall concentration and composition. Hundreds of differentially expressed probe sets between elongating and postelongation stem segments showed similar patterns of gene expression in the model legume and cultivated alfalfa. Differentially expressed genes included genes with putative functions associated with primary and secondary cell wall biosynthesis and growth. Mining of public microarray data for coexpressed genes with two marker genes for secondary cell wall synthesis identified additional candidate secondary cell wall-related genes. Coexpressed genes included protein kinases, transcription factors, and unclassified groups that were not previously reported with secondary cell wallassociated genes. M. truncatula has been recognized as an excellent model plant for legume genomics. The stem tissue transcriptome analysis, described here, indicates that M. Electronic supplementary material The online version of this article (doi:10.1007/s12155-009-9034-1) contains supplementary material, which is available to authorized users. M. Tesfaye : K. A. VandenBosch (*) Department of Plant Biology, University of Minnesota, 250 Biosciences Center, St. Paul, MN 55108, USA e-mail: [email protected] S. S. Yang : J. F. S. Lamb : H.-J. G. Jung : D. A. Samac : C. P. Vance : J. W. Gronwald Plant Science Research Unit, USDA-ARS, St. Paul, MN 55108, USA

truncatula has utility as a model plant for cell wall genomics in legumes in general and shows excellent potential for translating gene discoveries to its close relative, cultivated alfalfa, in particular. The natural variation for stem cell wall traits in Medicago may offer a new tool to study an expanded repertoire of valuable agronomic traits in related species, including woody dicots in the eurosid I clade. Keywords Medicago truncatula . Alfalfa . Transcript analysis . Genomics . Stems

Introduction Plant cell wall characteristics strongly affect the availability of lignocellulosic-derived sugar for fermentation and are a major factor affecting cost and efficiency of biomass conversion to biofuels, due to the challenges of pretreatment steps [8, 34, 43]. Arabidopsis, as the primary model plant, has provided a research platform for important discoveries of genes and gene functions associated with primary and secondary cell wall biosynthesis. The genomic tools available for Arabidopsis have also been used to identify genes involved in xylem formation for application in understanding wood formation (e.g., [44, 68]). Nevertheless, it is not clear whether Arabidopsis will provide all the tools necessary for an expanded repertoire of agronomic traits of value in crop species. For instance, previous genetic analysis and transcript profiling studies suggest a role for specific fasciclin-like genes in both primary and secondary wall formation [39, 46, 55]. However, many fasciclins that are highly expressed during formation of cellulose-rich tension wood in Populus spp. appear to lack orthologs in Arabidopsis [2, 39].


Legumes have many traits that make them attractive bioenergy crops, especially as components of mixed grass swards or in crop rotations with maize. Alfalfa (Medicago sativa) is a potential bioenergy legume that fixes atmospheric nitrogen and produces leaf and stem coproducts: the leaf meal for livestock feed [14] and dried stems for conversion to syngas [15] and/or fermentation to ethanol [12]. A perennial crop with high biomass yields, alfalfa is the fourth most widely grown crop in the USA [5]. Nevertheless, studying alfalfa is challenging because it is a cross-pollinated autotetraploid, with complex segregation and inheritance patterns. Because of its ease of genetic manipulation and small genome size, barrel medic (Medicago truncatula) has become a model species for genomic studies of the Fabaceae, including alfalfa. In contrast to alfalfa, M. truncatula is a lesser-grown annual, diploid, and selfpollinating species. Comparative mapping among many legumes has shown a high degree of conservation of gene content and gene arrangement [9, 70], as well as a very high degree of DNA sequence homology between alfalfa and M. truncatula [60]. In previous research, four M. truncatula accessions and two alfalfa genotypes were evaluated for stem tissue morphology and cell wall characteristics to ascertain whether M. truncatula displays comparable diversity in stem cell wall traits to alfalfa [54]. One obvious morphological difference between M. truncatula and alfalfa plants relates to their stem growth habit. Perennial alfalfa plants each year produce erect stems, while the annual barrel medic forms decumbent stems. Nevertheless, cross sections of M. truncatula and alfalfa stems showed similar patterns of tissue differentiation and growth [54]. During primary growth in alfalfa, deposition of nonlignified primary walls predominates in elongating stem internodes proximal to the apical meristem. In older stem internodes of alfalfa undergoing secondary growth, synthesis of lignin- and cellulose-rich secondary wall predominate due to deposition of secondary tissues by vascular cambium [18]. During the postelongation phase, xylem vessel element and fiber cells develop lignified primary and thickened, lignified secondary walls soon after differentiation from the cambium. Phloem fiber cells also develop a thickened cellulose-rich secondary wall, but only the primary wall of phloem fibers lignifies [18]. Similarly, the range of stem cell wall composition and content among M. truncatula accessions was found to resemble that of alfalfa [54]. Statistically significant differences in cell wall composition among the four M. truncatula accessions tested indicates that naturally occurring variation in M. truncatula may be a rich resource for discovering mechanisms regulating cell wall biosynthesis. Overall, previously published results suggest that analysis of plant cell wall traits in alfalfa and other legumes would

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be facilitated by evaluation of M. truncatula, with welldeveloped genetic and genomic resources [10, 64, 67]. In Arabidopsis, secondary cell wall formation was shown to increase with increasing distance from the shoot apical meristem toward the base of the inflorescence stem [62]. Sampling of stem segments along this developmental gradient has been instrumental in uncovering plant genes responsible for cell wall biogenesis and control in Arabidopsis [7, 17]. Prassionos et al. [49] used a similar approach for sampling stem segments of hybrid aspen for transcriptome profiling. Transcript analysis of woody plants has unveiled genes involved in lignin, pectin, and cellulose biosynthesis [29, 49]. Macroarray analysis of different plant organs and stem segments has also been used to profile transcript expression patterns of cell walls in maize [26]. These efforts have uncovered many cell wall-associated genes that have putative functions in the phenylpropanoid pathway, several transcription factor (TF) gene families, cell death proteins, and transporters, among others. Additionally, proteome analysis of plant cell walls has allowed the identification of cell wall-localized proteins that have not been previously identified using transcript profiling [35, 65]. The Affymetrix Medicago genome array [1], which contains more than 52,000 probe sets from barrel medic and alfalfa, has been instrumental in the identification of biologically meaningful gene expression patterns in M. truncatula [3, 31, 60] and M. sativa [60]. In this study, we used the Affymetrix Medicago array for a genome-wide expression study in young (elongating) and old (postelongation) stem segments of the M. truncatula accessions A17 and DZA315.16 (hereafter referred to as DZA) and alfalfa clones 252 and 1283. These germplasms were chosen because they express divergent cell wall composition. Identification of differential expression profiles between stem developmental stages was instrumental in identifying genes with putative functions in primary and secondary cell wall biosynthesis and growth in the model legume and cultivated alfalfa.

Methods Plant Culture Alfalfa and M. truncatula plants were grown in greenhouse and controlled growth chambers, respectively. Alfalfa clones 252 and 1283, which have been identified with consistent differences in stem cell wall cellulose and Klason lignin concentrations (Lamb and Jung, unpublished), were propagated from vegetative cuttings and grown in plastic pots (10×10×10 cm) containing soil/sand (1:1; v/v) in a greenhouse. When plants reached the full flower stage of development, alfalfa plants were cut back by removing the

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aerial herbage at 2-cm cutting height. Plants were allowed to regrow for approximately 6 weeks after cutting until they developed multiple stems. Stem segments were sampled at the late bud stage of development as described below. There were three replicates with 16 plants in each replicate. Plants were watered daily with tap water and fertilized weekly with water soluble fertilizer (20:10:20; N/P/K). For the M. truncatula experiment, seeds of M. truncatula A17 and DZA were scarified with sand paper and pregerminated in Petri plates on moist Whatman filter paper for 3 days at 4°C and then moved to room temperature for 24 h. Germinated seeds with approximately equal radicle lengths were planted in pots (10×10×10 cm) containing Metro-mix 200 (Sun Gro Horticulture, Bellevue, WA, USA) and were grown in a growth chamber (light intensity of 300 μmol m−2 s−1, temperature cycle of 25°C and 21°C, light and dark, with a 16-h photoperiod). One week after planting, seedlings were thinned to a single plant in each pot. Plants were watered with tap water as needed and fertilized weekly with water soluble fertilizer (20:10:20; N/P/K). Stem tissues were collected at 8 weeks after planting, when plants had developed multiple stems (three to four stems on each plant) with approximately eight to ten internodes per stem. There were three biological replicates with 21 pots in each replicate. Stem Tissue Harvest The transition from elongating to postelongation stage of stem internode development is easily identifiable in both M. truncatula and alfalfa by differences in pliability and suppleness of stem internodes. Stiff internodes are located lower on the stem axis and very pliable internodes near the tops of the stems. After identifying the internode which was in transition between these two developmental stages, it was excised and discarded. For microarray analysis, two internodes located immediately above (young stem segments, elongating) and below (old stem segments, postelongation) the transition internode were harvested. In general, young stem segments used for microarray analysis consisted of stem segments of the first and second internodes from the shoot apical meristem. Older stem segments generally contained the fifth and sixth internodes from the shoot apical meristem. Stem segments were immediately frozen in liquid nitrogen and stored at −80°C for subsequent RNA extraction. The remaining stem portions from each alfalfa plant (stem segments below the postelongating stem segment) were immediately collected and dried at 60°C for determination of cell wall composition [66]. RNA Extraction and GeneChip Hybridization Approximately 150 mg of stem tissue ground in liquid nitrogen was used for total RNA extraction using TRIZOL


reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. During the RNA extraction, contaminating genomic DNA was removed by incubating samples with RQ1 DNase following standard procedures suggested by the supplier (Promega, Madison, WI, USA). Ten micrograms of total RNA was used to produce biotin-labeled cRNA using Affymetrix kits following the manufacturer’s suggested procedures for eukaryotic reactions (Affymetrix, Santa Clara, CA, USA). Fifteen micrograms of biotin-labeled cRNA, fragmented as suggested by Affymetrix, was hybridized to the GeneChip® Medicago Genome Array. The integrity and quality of total RNA and fragmented biotinlabeled cRNA were verified using the Agilent 2100 Bioanalyzer RNA 6000 Nano LabChip (Agilent Technologies, Santa Clara, CA, USA). GeneChips were hybridized, washed, stained, and scanned as previously described [60]. Microarray Data Analysis In all of the data analyses, gene expression signals corresponding to the bacterial microsymbiont probe sets were excluded. Gene expression values were calculated with the robust multi-array average [33] using quantile normalization, as provided with the Genedata Expressionist Pro version 4.5 (Genedata, San Francisco, CA, USA). Presence or absence calls of expression data for each probe set were made using MAS5 [42]. Principal components analysis (PCA) was initially used to evaluate gene expression patterns between young and old stem internodes of M. truncatula and alfalfa. PCA was conducted using the Genedata Expressionist Pro version 4.5 platform (Genedata). Statistical analysis of the stem microarray data between young and old stem segments was based on the t test (p
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